Abundant circumstantial evidence indicates that oxidative stress contributes to many consequences of normal aging and several major diseases, including cardiovascular diseases, diabetes, neurodegenerative diseases, and cancer. Oxidative stress is generally defined as an imbalance of prooxidants and antioxidants. However, despite a wealth of scientific evidence to support increased oxidative tissue damage, large-scale clinical studies with antioxidants have not demonstrated significant health benefits in these diseases. One of the reasons may be due to the inability of the available antioxidants to reach the site of prooxidant production. The mitochondrial (mt) electron transport chain (ETC) is the primary intracellular producer of ROS, and mt themselves are most vulnerable to oxidative stress. Protecting mt function would therefore be a prerequisite to preventing cell death caused by mt oxidative stress. The benefits of overexpressing catalase targeted to mt (mCAT), but not peroxisomes (pCAT)(see Project 1), provided proof-of-concept that mt-targeted antioxidants would be necessary to overcome the detrimental effects of aging (9-12). However, adequate delivery of chemical antioxidants to the IMM remains a challenge. The common approach for mt targeting makes use of the potential gradient across the IMM. The triphenylalkyl-phosphonium cation (TPP*) has been conjugated to lipophilic antioxidants such as Vit E (MitoE) and coenzyme Q (MitoQ)(13), and plastoquinone (SkQI) (14) to facilitate their delivery into the mt matrix. However, it is uncertain whether these TPP+ -conjugated antioxidants will be adequately taken up by stressed or aged mt. Furthermore, accumulation of these lipophilic cations in the mt matrix can disrupt mt potential and inhibit mt respiration and ATP production (15). In addition, mt redox cycling of MitoQ can lead to superoxide production and cellular apoptosis (16,17). Thus it is essential to develop alternate mechanisms for targeting therapeutic agents to mt. In a chance discovery, Szeto and Schiller found that a series of cell-permeable aromatic-cationic tetrapeptides (SS peptides) selectively target mt and concentrate 1000-fold in the IMM(4). Although the exact mechanism by which these peptides target the IMM is not understood, it is clear that their uptake is not dependent on mt potential(4). Mt targeting does appear to be dependent on the alternating aromatic-cationic amino acid motif in these peptides, and this has been confirmed by other investigators(8). Significantly, the SS peptides represent the first class of chemical agents to target the IMM rather than mt matrix. These aromatic-cationic peptides can be used to facilitate the delivery of other cargoes to mt (18), and can therefore be conjugated to available antioxidants. More importantly, antioxidant properties can be incorporated into this aromatic-cationic motif simply by the selection of appropriate aromatic and cationic amino acid residues. One of the SS peptide analogs, SS-31, possesses intrinsic antioxidant ability because the modified tyrosine residue is redox-active and can undergo one-electron oxidation(19). We have shown that SS-31 can neutralize H2O2, hydroxyl radical, and peroxynitrite, and inhibit lipid peroxidation (4,6). SS-31 is at least 100-fold more potent than the TPP+ -conjugated antioxidants in protecting cultured cells from proxidants such as t-butyl-hydroperoxide (20) and hypochlorous acid (5), and can even confer protection after mt depolarization(5). The SS peptides were designed to be stable against peptidase degradation and have excellent pharmacokinetic properties(21). SS-31 has demonstrated remarkable efficacy in animal models of ischemia-reperfusion injury(22,23), neurodegenerative diseases (24,25), and metabolic syndrome(11). SS-31 can readily cross the BBB and toxicology studies have revealed an excellent safety profile for SS-31 {Stealth Peptides Inc, personal communication). The therapeutic potential of the SS peptides has been discussed in several reviews(26-28) (N.B. Cornell Research Foundation has licensed the SS peptide technology platform to Stealth Peptides for clinical development). Significantly, SS-31 provided comparable protection as mt catalase overexpression in three animal models of mt oxidative stress (see Approach below), thus supporting our proposal that this small molecule chemical approach can be used to complement our genetic approach in studying the role of mt oxidative stress in aging and health span. The proposed Chemistry Core will carry out large-scale synthesis of SS-31 and SS-20 to provide to all projects for the initial proof-of-concept studies. At the same time, we will proceed to design an orally-active analog of SS-31 for the chronic aging studies. In addition to providing SS-31, we also propose to further investigate the use of this novel IMM-targeting strategy to develop other antioxidants with different mechanisms of action. Mt oxidative stress is commonly defined as a disturbance in the prooxidant-antioxidant balance, implying that interventions should be based on either reducing the level of prooxidants or the addition of appropriate antioxidants. Alternatively, it was recently proposed that mt oxidative stress may be defined as "a disruption of electron transfer reactions leading to an oxidant/antioxidant imbalance...(29). The ETC can be viewed as a high-flux electron transfer pathway, and inhibition of electron transfer in the ETC or redox cycling agents greatly simulate ROS generation(30). The view that mt oxidative stress is a disruption of redox circuitry would imply that it may be more fruitful to develop strategies to improve electron flow by facilitating electron transfer. The proposed Chemistry Core will extend the design of the mt-targeted peptides to incorporate and enhance one or more of the following modes of action: (i) scavenging excess ROS, (ii) reducing ROS production by facilitating electron transfer, or (iii) increasing mt reductive capacity. The advantage of peptide molecules is that it is possible to incorporate natural or unnatural amino acids that can serve as redox centers, facilitate electron transfer, or increase sulfydryl groups while retaining the aromatic-cationic motif required for mt targeting. The proposed design strategies are supported by known electron chemistry and will be confirmed by chemical, biochemical, cell culture, and animal studies. State-of-the-art physical, chemical and molecular biology approaches will be used to screen the new analogs for mt ROS production and redox regulation, testing and validating the hypothesized molecular modes of action. The most promising analogs will be provided to the various projects for evaluation in mt, cellular, and tissue models. The proposed studies represent a novel integrated approach to the design of mt-targeted antioxidants that is significantly different from other efforts in the field.